New insights into the stipitate hydnoid fungi Sarcodon, Hydnellum, and the formerly informally defined Neosarcodon, with emphasis on the edible species marketed in Southwest China

Sarcodon and Hydnellum are two ectomycorrhizal genera of important ecological and economic value in Southwest China, and they are common in the free markets in this region. It was estimated that more than 1,500 tonnes of them were sold as edible per year, but there was little information about the taxonomic placements of these edible mushrooms sold in the markets. Traditional concepts of the two genera have also been challenged recently, and circumscription of Sarcodon and the informally defined clade “Neosarcodon” remained unresolved. In the present study, specimens collected in the field and purchased from the markets in Southwest China were analyzed based on morphological characters and DNA sequences. Phylogeny of the traditional Sarcodon s. lat. and Hydnellum s. lat. was reconstructed from the combined internal transcribed spacer (ITS), nuclear large ribosomal subunit (nLSU) and RNA polymerase II second largest subunit (RPB2) dataset based on expanded samples to reevaluate the taxonomic placements of the two genera. In the present molecular analyses, four distinct clades were recovered and strongly supported: Hydnellum, Neosarcodon, Phellodon and Sarcodon. Neosarcodon is formally introduced as a generic name to include nine species previously placed in Sarcodon, and the delimitation of Sarcodon is revised based on phylogenetic and morphological studies. Phylogenetic analyses also revealed an unexpected species diversity (17 phylogenetic species) of Sarcodon and Hydnellum in the markets; nine phylogenetic species of Sarcodon and eight of Hydnellum were uncovered from the samples collected in the markets. Eight species were resolved in the traditional S. imbricatus complex, with S. imbricatus s.str. being the most common edible stipitate hydnoid fungal species. Three of the edible Hydnellum species (H. edulium, H. subalpinum, and H. subscabrosellum), and five separated from the S. imbricatus complex (Sarcodon flavidus, S. giganteus, S. neosquamosus, S. nigrosquamosus, and S. pseudoimbricatus), are described as new. Three new Chinese records (H. illudens, H. martioflavum, and H. versipelle), and the notable S. imbricatus and S. leucopus are also reported.


INTRODUCTION
Sarcodon and Hydnellum are common and important ectomycorrhizal (ECM) genera that traditionally differ in basidiome structure and are placed in Bankeraceae.These are stipitate hydnoid genera that both have brown tinted basidiospores, while Sarcodon has softer and fleshier basidiomata and Hydnellum ones that are hard and dry.The identification of Sarcodon and Hydnellum species was traditionally mainly based a limited range of morphological characters, such as the color of the pileus and stipe, the arrangement of the pileus surface, organoleptic features (smell and taste), and spore size (Harrison 1964(Harrison , 1984;;Maas Geesteranus and Nannfeldt 1969;Maas Geesteranus 1971, 1975;Baird 1985;Baird and Khan 1986;Harrison and Grund 1987;Stalpers 1993;Pegler et al. 1997;Strid 1997).However, in the last decade, DNA sequence analyses have shown the traditional generic delimitation of the two genera to be somewhat questionable (Nitare and Högberg 2012;Baird et al. 2013;Miscevic 2013;Loizides et al. 2016;Vizzini et al. 2016).Larsson et al. (2019), based on nuclear large ribosomal subunit (nLSU) sequence analysis, showed that Hydnellum and Sarcodon were distinct genera but that the current division based on basidioma texture made Sarcodon paraphyletic with respect to Hydnellum.Consequently, some species of Sarcodon were moved to Hydnellum.Additionally, a distinct clade, "Neosarcodon", was informally defined in the internal transcribed spacer (ITS) sequence analysis of that study.Mu et al. (2021) also confirmed that the traditional concept of Sarcodon was not monophyletic, and their previously described new species of Sarcodon (Mu et al. 2020) were shown to be nested in Hydnellum.Notably, all the known species in the traditional Sarcodon sect.Scabrosi were confirmed to belong in Hydnellum, and the new subgenus Hydnellum and subgen.Scabrosum was established to accommodate these species (Mu et al. 2021).The traditional concept of Sarcodon is therefore much reduced with less than 50 species retained, but the phylogenetic relationships and monophyly of the remaining Sarcodon species have not been resolved using phylogenetic analyses (Larsson et al. 2019;Mu et al. 2021).Further studies based on a more extensive sampling and an expanded dataset are needed to develop more robust generic limits for these fungi.
Although members of the stipitate hydnoid fungal genera have been considered endangered and included in the Red Data Lists of several European countries (Hrouda 1999(Hrouda , 2005;;Walleyn and Verbeken 2000;Nitare 2006;Senn-Irlet et al. 2007), they are of important economic value in Southwest China, especially Sarcodon species, and are common in the free markets in this region (Fig. 1).It is estimated that more than 1,500 tonnes of Sarcodon species are sold in the free markets per year in Sichuan province.Studies in recent years have demonstrated the high species diversity of the stipitate hydnoid genera Hydnellum and Phellodon in China (Mu et al. 2020(Mu et al. , 2021(Mu et al. , 2022b;;Song et al. 2022a).However, little taxonomic information about Chinese Sarcodon was involved in these studies.In Southwest China, Sarcodon species with the pileus covered by prominent squamules were usually called "hei hu zhang" or "zhang zi jun" in the markets, and were commonly identified as S. imbricatus, S. aspratus or S. squamosus (Dai and Li 1994;Li et al. 2015;Yang et al. 2021), among which S. imbricatus was the most widely used name.Although these "hei hu zhang" look the same at first glance, however, we found that these "S.imbricatus" specimens exhibited some subtle differences when examined closely and could represent different taxa.Molecular analyses in the laboratory confirmed this speculation.
Hydnellum is also common in the markets, but much less so than Sarcodon.Sometimes, Hydnellum basidiomes were found to be mixed together with "S.imbricatus" (Fig. 1b) and were thought to be Sarcodon in the traditional sense; however, these specimens actually belonged to Hydnellum based on comprehensive morphological and molecular analyses.In some regions of Southwest China, Hydnellum species were separated from Sarcodon s.str.and were called "jia hu zhang jun (pseudo-Sarcodon)" in the markets.Moreover, little information about the taxonomic placements of these Hydnellum species sold in markets was documented in China.Mu et al. (2020Mu et al. ( , 2021) ) described 13 new Hydnellum species from China, most of which seemed to be different from the edible Hydnellum species sold in the markets.In a review of the diversity of Chinese macrofungal resources (Wu et al. 2019), only three "Sarcodon" species (S. imbricatus, S. leucopus, S. violaceus) and two Hydnellum species (H.concrescens and H. cumulatum) were listed as edible; S. violaceus has been considered as Bankera violacea.In other words, prior to the present study, only two edible Sarcodon species and two edible Hydnellum species were recorded in China.Additionally, it was unclear whether the names applied to these edible stipitate hydnoid fungi were correct.
In the present study, edible Sarcodon and Hydnellum specimens purchased from the markets in Southwest China, and specimens collected in the field were subjected to morphological and molecular analysis to clarify the situation.Furthermore, the phylogenetic relationships of the traditional genera Hydnellum and Sarcodon were reevaluated based on the combined ITS, nLSU and RNA polymerase II second largest subunit (RPB2) sequences, and the generic limits of Sarcodon s.lat.were revised based on morphological and molecular analyses.

Morphological descriptions
Fresh specimens were collected in the field or markets, and our macro-morphological descriptions were based on fresh materials.The color notations followed Kornerup and Wanscher (1978).Basidiospores, basidia, and pileipellis were mounted and measured in 5% KOH, and observed using a Leica DM5000B microscope.Basidiospores were measured with ornamentation in profile view.All holotype collections are kept in the Fungarium of Sichuan Academy of Agricultural Sciences (SAAS).

DNA extraction, polymerase chain reaction PCR amplification and sequencing
The procedures used to conduct genomic DNA extraction, PCR amplification, and sequencing were the same as in our previous study (He et al. 2013).The primers used for the ITS regions were ITS5 and ITS4 (White et al. 1990), the primers used for RPB2 amplification were rpb2-6f and rpb2-7r (Liu et al. 1999), and the nLSU regions were amplified using the primer pair LR0R and LR5 (http:// www.biolo gy.duke.edu/ fungi/ mycol ab/ prime rs.htm).The PCR products were sequenced in both directions.

Sequence alignment and phylogenetic analyses
The published sequences of Sarcodon and Hydnellum downloaded from GenBank were carefully checked; those of low quality and suspect were excluded.Sequences newly generated in this study were manually corrected according to the sequence chromatograms.Phylogenetic analyses were performed using Maximum Likelihood (ML) and Bayesian analysis based on the combined ITS, nLSU, and RPB2 dataset; Amaurodon aquicoeruleus and A. viridis were used as the outgroups following Mu et al. (2021).The sequences used in this analysis are listed in Table 1 and aligned in muscle 3.6 (Edgar 2004).If necessary, the aligned sequences were manually modified employing Mega 11 (Tamura et al. 2021).All sequence data generated for this study can be accessed via Gen-Bank: https:// www.ncbi.nlm.nih.gov/ genba nk/.All alignments for phylogenetic analyses have been deposited in TreeBASE (http:// purl.org/ phylo/ treeb ase/ phylo ws/ study/ TB2: S30772).Single-locus phylogenies were constructed to detect incongruence among individual genes using the maximum likelihood (ML) method.As no conflicts were detected among the well-supported clades, the sequences of ITS, nLSU and RPB2 were combined for further analyses.
ML analyses were carried out by the web RAxML Version 8 http:// www.phylo.org/ sub_ secti ons/ portal/) under the GTR + G + T model with 1000 bootstrap replicates (Miller et al. 2010;Stamatakis 2014).The "Find best tree using maximum likelihood search" option was selected when analysing.Bayesian analysis was performed using MrBayes 3.2.7 (Ronquist and Huelsenbeck 2003).The best substitution models for each marker were selected by using the Akaike Information Criterion (AIC) in jModelTest 2. 1.10 (Darriba et al. 2012).The RPB2 region was treated as a single partition because that there was no introns in the studied sequences.The GTR + I + G model was selected for ITS, TIM1 + I + G for nLSU, and TrN + I + G for RPB2.Four simultaneous Markov chains were run starting from random trees, keeping one tree every 1000th generation until the average standard deviation of split frequencies was below 0.01.The burn-in value was set to discard 25% of trees when calculating the posterior probabilities.The Bayesian posterior probabilities were obtained from the 50% majority rule consensus of the trees kept.FigTree v1.4.4 (Rambaut 2018) was used to display the resulting trees.

Molecular analyses
The analysis included 275 sequences representing 82 taxa in our analysis; 121 sequences were generated in the present study (47 ITS, 38 nLSU, and 36 RPB2 sequences).After trimming, 2763 characters were retained in the dataset, including 1140 for ITS, 946 for nLSU, and 677 for RPB2.The phylogenetic construction performed with ML and Bayesian Inference (BI) analyses for the combined dataset showed similar topology, and only the ML tree is shown in Fig. 2.
In the combined analyses, four distinct clades were recovered: Hydnellum, Neosarcodon, Phellodon and Sarcodon, all of which were strongly supported in both the ML and Bayesian analyses.Species in the Neosarcodon clade possessed the characters of an adnate hymenophore, an indistinct odor, and the absence of hymenial cystidia.Molecular analyses further confirmed this as a distinct group that could be treated as a genus.
The molecular analyses showed that at least 17 phylogenetic species of Sarcodon and Hydnellum were found in the markets in Southwest China: nine Sarcodon species (indicated by black stars ★ in the tree, Fig. 2) and eight of Hydnellum species (indicated by black triangles ▲ in the tree, Fig. 2).Eight of the nine edible Sarcodon species were grouped in the S. imbricatus complex, and the other as S. leucopus.Except for S. imbricatus and S. leucopus, the other Sarcodon species (S. flavidus, S. giganteus, S. neosquamosus, S. nigrosquamosus, and S. pseudoimbricatus) sold in the markets formed distinct clades that were different from the known species.Sarcodon giganteus, S. nigrosquamosus, and S. pseudoimbricatus clustered in the same clade, suggesting a close relationship.In

Taxonomy
Based on the molecular and morphological evidence, we formally propose the clade "Neosarcodon" as defined by Larsson et al. ( 2019) as a distinct genus, Neosarcodon; the corresponding species should be moved from Sarcodon to Hydnellum.Accordingly, the genus description of Sarcodon should be revised.The distinct groups in Hydnellum will be redefined when more data become available.Diagnosis: Differs from Hydnellum fagiscabrosum in the lack of a contrasting whitish pileal margin.
Description: Pileus 5-9 cm diam., slightly plano-convex to planar, depressed in the center; surface brownish yellow to reddish brown; cracked by fissures forming large scales in the center, becoming depressed and small scales toward margin; scales arranged somewhat concentrically, brownish yellow to brown.Spines decurrent, whitish when young, pale gray to pale brownish gray when mature, becoming brown when touched, up to 0.8 cm long, spine tips whitish.Stipe 4-6 cm in length, 1-1.5 cm diam., paler than the pileus, becoming pale brownish when touched, with a distinctive bluish green color within the base of the stipe, slightly eccentric to central, cylindrical to attenuate below, nearly glabrous, covered by short spines in the upper stipe, solid.Context whitish.
Ecology and distribution: Solitary on the ground in forests dominated by Pinus and Quercus.
Remarks: Hydnellum fagiscabrosum is similar to H. edulium, but differs in the contrastingly whitish pileal margin.H. edulium and H. illudens are similar morphologically, however, H. illudens is usually found at higher elevations (> 3000 m) in subalpine areas while H. edulium is mainly distributed in subtropical forests at lower elevations (< 3000 m).DNA sequence analyses show that H. scabrosum and H. illudens are rather distant from H. edulium.
Additional specimens examined: China: Sichuan Province: Panzhihua City, purchased from the free market, 16 Aug.2017, He (SAAS 2870); Guangyuan City, Lizhou District, Tianzhao Mountain, Sheli Tower, 14 Aug.Description: Basidiomata rarely single, gregarious to concrescent.Pileus 5-20 cm broad from fused pilei, planar to convex or depressed at disc, imbricate from multiple pileoli, concentric zones near margin, margin irregular to lobed from fused pilei, surface irregular and rarely smooth, generally radially rugulose to rugose, spongy tomentose to tomentose with fibrillose hairs, becoming matted or pitted; usually white nearest margin even when old, brownish yellow to brown near center.
Spines decurrent, up to 8 mm long, crowded, concolorous with pileus to brown in age.Stipe 1.5-6 cm in length, 0.8-2 cm in diam., terete to subattenuate below, surface irregular from indeterminate growth, concolorous with pileus flesh.Context up to 1 cm nearest stipe, concolorous with pileus.Taste mild to none; odor not distinct.
Ecology and distribution: Scattered, gregarious to concrescent in forests dominated by Abies.
Remarks: Multiple fused basidiomata are diagnostic for this species.This species is most similar to H. cumulatum by the fused and brown basidiomata.However, H. cumulatum differs by bruising black, and in its occurrence in pine forest.H. spongiosipes is also similar, but differs in its larger basidiospores (6-7 × 5-6 μm, Baird et al. 2013).
Additional Description: Pileus 3-7 cm broad, plano-convex with a central depression, brownish yellow to reddish brown around the center, paler near margin; covered with floccose scales, almost smooth at margin.Spines strongly decurrent, up to 5 mm long, crowded, at first whitish, becoming yellowish brown when mature.Stipe 3.5-5 cm in length, 0.8-1.5 cm in diam., slightly eccentric to central, cylindrical to attenuate below, covered by short spines in the upper stipe, concolorous with the pileus, at the base bluish-grey or blackish-green, solid.Flesh pale yellow.Smell and taste not distinct.
Ecology and distribution: Solitary on the ground under Pinus and Abies.
(Figs. 1f, 4h, i) Description: Pileus 6-10 cm, plano-convex to almost planar, somewhat depressed above the stipe, cracked by fissures forming somewhat pointed upward scales in depression, becoming small and depressed scales toward margin, ochraceous to fulvous brown, sometimes darker in the middle.Spines decurrent, up to 5 mm long, crowded, at first whitish, then becoming grayish brown when mature.Stipe 3-5 in length, 1-2.5 mm in diam., above concolorous with the pileus, at the base greyish blue, eccentrical to central, tapering downwards with a short rooting point, solid.Flesh pale greyish.Smell not distinct, taste slightly bitter.
Ecology and distribution: Solitary on ground under Quercus.
Remarks: H. illudens, known from Europe, is common in the free markets of Aba Tibetan and Qiang Autonomous Prefecture.Basidiospores of the Chinese collections are larger than those of European samples [4.7-5.7 (-6.1) × 3.5-4.5 µm, Nitare et al. 2021].However, the similarity of ITS sequences between them is up to 99.67%, showing that they are the same species.
Specimens (Fig. 1g, 4k) Description: Pileus up to 9 cm broad, plano-convex to nearly planar, often depressed in the center, margin irregularly shaped, sometimes concrescent, subvelutinous to fibrillose, pale yellow brown to light brown.Spines subdecurrent, up to 5 mm long, crowded, gray whitish to gray.Stipe 3-5 cm in length, 1-1.8 cm in diam., central to slightly eccentric, cylindrical or attenuate below, covered by short spines in the upper stipe, subtomentose to fibrillose squamose below, concolorous with pileus, solid.Context up to 1 cm thick, white.Taste and smell not distinct.
Ecology and distribution: Solitary on the ground under Abies and Pinus.
Remarks: ITS sequence analysis showed that these Chinese collections are H. martioflavum, and the basidiospores of Chinese materials are slightly larger than those of specimens collected from the United States [(4) 5-6 × 3-5 μm, Baird et al. 2013 (Fig. 4l).
Description: Pileus up to 20 cm broad, convex to planar or depressed, sometimes irregular, subsquamulose to fibrillose or pubescent, margin often lobed or irregular, brownish orange to reddish brown.Spines decurrent, up to 0.9 cm long, crowded, whitish when young, becoming darker when mature, white on tips.Stipe 4-8 cm in length, 0.8-1.2cm in diam., central to slightly eccentric, cylindrical, terete to attenuate below, subsquamulose, fibrillose, concolorous with pileus, solid.Context up to 1 cm thick nearest stipe, white.Taste and odor not distinct.
Ecology and distribution: Solitary on the ground under Abies.
Description: Pileus 7-12 cm in diam., plano-convex, depressed in the center; surface yellow, becoming brownish yellow when touched; cracked by fissures forming large and coarse scales in the center, becoming depressed small and floccose scales toward margin; scales arranged concentrically, brownish yellow to brown.Spines decurrent, whitish when young, pale gray to pale brownish gray when mature, becoming brown when touched, up to 0.8 cm long.Stipe 2.5-4 cm in length, 1-1.5 cm in diam., concolorous with the pileus, becoming pale brownish when touched, slightly eccentric to central, cylindrical to attenuate below, nearly glabrous, covered by short spines in the upper stipe, solid.Context pale yellowish.Taste mild; smell strong but agreeable when dried.
Ecology and distribution: Solitary, scattered or in small clusters on the ground in mixed forests of Abies, Pinus and Quercus.
Remarks: This species is distinguished by the yellowish pileus, which became brownish yellow when touched.Sarcodon flavidus is morphologically similar to S. imbricatus, but the latter differs in the darker pileus covered by imbricate and larger scales, as well as larger spores.S. scabrous and S. underwoodii are somewhat similar to S. flavidus morphologically; however, the two species have been proved to be members of Hydnellum.
Description: Pileus 12-40 cm in diam., fan-shaped, usually depressed above the stipe; surface grayish white to beige; cracked by fissures forming somewhat upward-pointed scales in depression, becoming small scales toward margin; scales brownish yellow, becoming brown when touched.Spines decurrent, grayish white when young, pale gray to brownish gray when mature, becoming brown when touched, up to 1.2 cm long.Stipe 7-10 cm in length, 1.5-2 cm in diam., concolorous with the pileus, becoming brown when touched, eccentric to lateral, cylindrical to attenuate below, nearly glabrous, covered by short spines in the upper stipe, solid.Context grayish white; taste mild; smell strong but agreeable when dried.
Ecology and distribution: Scattered or in small clusters on the ground in forests dominated by Pinus and Quercus.
Remarks: S. giganteus is distinguished from the other Sarcodon species by its rather large and paler basidiomata when mature.The pale and sparsely distributed scales are different from the most common S. imbricatus.However, the young S. giganteus is hardly to separate from the other Sarcodon species based on the morphological characters.ITS and RPB2 sequences of S. giganteus and the similar species are quite different.
Sarcodon neosquamosus Xiao L. Description: Pileus 6-11 cm in diam., plano-convex to nearly planar, not depressed or slightly depressed in the center; surface reddish brown; covered by coarse scales with suberect tips, becoming small depressed floccose scales toward the margin; scales concolorous with the background at first, becoming dark brown with age.Spines usually short decurrent, grayish white when young, pale gray when mature, becoming brown when touched, up to 0.6 cm long.Stipe 3-5 cm in length, 0.8-1.4cm in diam., pale grayish, becoming brownish when touched, usually eccentric, cylindrical and somewhat inflated at the base, solid.Context whitish to grayish white; taste mild; smell strong but agreeable when dried.
Ecology and distribution: Solitary on the ground under Pinus.
Remarks: S. squamosus is the most similar species to S. neosquamosus, and it is difficult to separate the two species based on the pileal morphological characters alone.According to Johannesson et al. (1999), S. squamosus has a yellowish brown to vinaceous brown pileus, the spines are often with a tint of greyish blue when fresh, the stipe is attenuated at the base, and the context is whitish but sometimes blackish brown in the stipe base.Additionally, the partial LSU sequences and complete ITS sequences of S. squamosus, published by Johannesson et al. (1999), Vizzini et al. (2013) and Larsson et al. (2019), are different from those of S. neosquamosus.S. imbricatus is separated from S. neosquamosus by its brown pileus with a more prominent center depression, as well as its occurrence in spruce forest.
Diagnosis: Differs from Sarcodon imbricatus by having relatively larger pileus, dark smaller and denser pileal scales, and relatively slender stipe.
Description: Pileus 8-20 cm in diam., plano-convex to nearly planar, depressed in the center; surface whitish gray to pale brown; cracked by fissures forming large and coarse scales with tips pointed upward in the center, becoming small depressed floccose scales toward the margin; scales concolorous with the background at first, becoming darker with age, tips often brown to black when mature.Spines decurrent, grayish white when young, pale gray to brownish gray when mature, becoming brown when touched, up to 0.7 cm long.Stipe 3-5.5 cm in length, 1-1.6 cm in diam., pale brownish yellow to concolorous with the pileus, eccentric to central, occasionally lateral, cylindrical to attenuate below, covered by short spines in the upper stipe, solid.Context grayish white; taste mild; smell strong but agreeable when dried.
Ecology and distribution: Solitary on the ground under Pinus and Quercus.
Remarks: Sarcodon nigrosquamosus is separated from S. imbricatus by having relatively larger pileus, smaller and denser pileal scales and a relatively slender stipe.S. aspratus and S. squamosus were usually treated as synonyms of S. imbricatus, however, several studies based on ITS sequences have shown that they are different species (Johannesson et al. 1999;Vizzini et al. 2013).According to Johannesson et al. (1999) Description: Pileus 7-15 cm in diam., plano-convex to nearly planar, depressed in the center; surface whitish; cracked by fissures, forming large and sparse scales with tips pointed upward in the center, becoming small scales toward the margin;scales concolorous with the background at first, tips often darker when mature.Spines strongly decurrent, grayish white to pale gray, becoming brownish when touched, up to 0.7 cm long.Stipe 3-5 cm in length, 1-1.3 cm in diam., concolorous with the pileus, eccentric, occasionally lateral, cylindrical to attenuate below, covered by short spines in the upper stipe or almost the entire stipe, solid.Context grayish white; taste mild; smell strong but agreeable when dried.
Ecology and distribution: Growing in small clusters or solitary on the ground in forests dominated by Pinus.
Remarks: S. imbricatus is the most similar species to S. pseudoimbricatus.However, S. pseudoimbricatus is separated from S. imbricatus by having a relatively paler pileus and scales.
(Figs. 7a-c) Description: Pileus 6-22 cm in diam., plano-convex, sometimes irregular, depressed in the center; surface brownish yellow; covered by coarse scales with tips suberect; scales concolorous with the background at first, becoming dark brown with age.Spines decurrent, grayish white when young, pale gray when mature, becoming brown when touched, up to 0.9 cm long.Stipe 2.5-6 cm in length, 0.8-1.6 cm in diam., pale grayish, becoming brownish or greenish when touched sometimes, usually eccentric, cylindrical to attenuate below, solid.Context grayish white; taste mild; smell strong but agreeable when dried.
Ecology and distribution: Solitary or scattered on the ground under fir-spruce forests.
Remarks: According to the identification of 132 specimens, S. imbricatus is the most common hydnoid species in the free markets.Examination of macromorphological characters showed that some collections of S. imbricatus staining greenish when touched (Fig. 7c).This character was not mentioned in the previous literature, and we thought these collections to be a different species from S. imbricatus, however, molecular evidence proved that they are identical to S. (Figs. 1d, e, 7d-f ) Description: Pileus 6-20 cm in diam., plano-convex to nearly planar, sometimes irregular; surface grayish white, becoming somewhat grayish purple when touched; almost smooth, somewhat velutinous and chapped.Spines decurrent, grayish white when young, pale gray when mature, becoming brown when touched, up to

DISCUSSION
Sarcodon and Hydnellum species are common in markets in Sichuan and Yunnan Provinces in Southwest China, and they are of important economic value to the local people (Fig. 1).In addition, some species of the two genera, including S. imbricatus and S. leucopus, are reputed to have important medicinal functions (Ma et al. 2014;Tan et al. 2020).In the present study, the species diversity of the two genera marketed in Southwest China was analyzed based on morphological characters and DNA sequences (ITS, RPB2 and nLSU).Five species separated from the S. imbricatus complex and three of Hydnellum are described in this work as new; three new Chinese records of Hydnellum and the notable S. leucopus are also presented.
The molecular analyses strongly supported the "Neosarcodon" clade defined by Larsson et al. (2019), as well as the monophyly of the much reduced "Sarcodon" clade and the revised Hydnellum.According to the descriptions and illustrations of members in the Neosarcodon clade (Grupe et al. 2015(Grupe et al. , 2016)), Neosarcodon differs from Sarcodon in the adnate hymenophore, its indistinct odor, and the absence of hymenial cystidia.Based on the morphological and molecular evidence, Neosarcodon is formally introduced as a distinct genus here.Phylogenetic analyses also showed that the species diversity of Sarcodon and Hydnellum in the markets was much higher than previously thought, and at least 17 phylogenetic taxa of the two genera could be found in the free markets in Southwest China.Five (H.illudens, H. martioflavum, H. versipelle, S. imbricatus and S. leucopus) of the 17 species are shared with other continents.
Although Sarcodon is one of the most important wild edible mushrooms in Southwest China, it is rather difficult to find these mushrooms in the field for research due to their occurrence in subalpine forests and comprehensive collection by local people.Prior to this study, fewer than 50 taxa were included in the reduced concept of Sarcodon, and only two names (S. imbricatus and S. leucopus) were widely used in China.In the present molecular analyses, nine Sarcodon taxa were recovered in China, all of which could be found in the markets sold as edible (marked with black stars ★ in the tree).Surprisingly, seven taxa were included in the traditional S. imbricatus complex in addition to S. imbricatus: S. flavidus, S. giganteus, S. nigrosquamosus, S. peseudoimbricatus, and S. subsquamosus which are formally described here; as well as two unnamed Sarcodon species that have been temporarily shelved due to the lack of sufficient knowledge about these species.Among these edible Sarcodon species, S. leucopus can be easily separated from the others by its almost smooth pileus and rather bitter taste, and this species is rather popular in Yajiang County, Sichuan province.Members in the S. imbricatus complex are mixed together in the markets, where they are usually called "hei hu zhang" or "zhang zi jun".According to the present investigation and molecular analyses on 132 specimens collected in markets, the true S. imbricatus is actually the most commonly sold species in the markets, while S. giganteus is restricted to Liangshan Yi Autonomous Prefecture in Sichuan Province.Although these species are similar to S. imbricatus in macromorphology, the mature basidiomes exhibit several subtle but stable differences.S. nigrosquamosus has dense, small, and almost black scales; S. flavidus has a yellow pileal surface; the rather large basidiomata, grayish white pileus, and scales are distinguishing characters for S. giganteus; S. neosquamosus possesses a reddish brown pileus; and S. pseusoimbricatus is unique in its pale and sparse scales.However, the young basidiomes of S. giganteus, S. neosquamosus, S. nigrosquamosus, and S. imbricatus are all rather similar to each other and cannot be distinguished based on morphological characters alone.In the literature, S. squamosus and S. aspratus are always linked with S. imbricatus; in addition, they are often synonymized with S. imbricatus, and little information about them has been documented.From the limited information in the literature, it is known that S. squamosus grows in pine forests, and S. aspratus grows in Quercus forests (Johannesson et al. 1999;Vizzini et al. 2013).In the study of Vizzini et al. (2013), S. squamosus and S. aspratus were shown to be independent species distinct from S. imbricatus, and another species (S. quercinofibulatus) occurred under Quercus that was also separated from the S. imbricatus complex based on ITS sequences.Sequence analyses showed that S. quercinofibulatus and S. squamosus were different from the Chinese collections.In GenBank, only two ITS sequences (DQ448877 and AF335110) were labeled as S. aspratus, but these sequences were of low quality and were not included in the present analyses.Judging from the two sequences, they were also different from the Chinese collections.
Hydnellum is an important genus of stipitate hydnaceous fungi.H. concrescens and H. cumulatum were reported as edible in China (Wu et al. 2019), but the present analyses did not reveal their occurrence in China.A recent study (Mu et al. 2021) presents 11 new species of Hydnellum with woody basidiomata from China that are rarely found in markets.Hydnellum collections sold in the markets are usually more fleshy than other Hydnellum species, and many of them were thought to be Sarcodon s. lat. in the traditional sense.In the present investigation, eight Hydnellum species were found in the markets in Southwest China (marked with black triangles ▲ in the tree).Five of these edible Hydnellum species (H. edulium, H. illudens, H. lidongensis, H. subscabrosellum and H. grosselepidotum), are placed in the clade consisted of H. scabrosum and relatives (Hydnellum subgenus Scabrosum in Mu et al. 2021).All the known species in this clade were placed in Sarcodon prior to Larsson et al. (2019).The macromorphological characters of this group are more distant from the typical Hydnellum than Sarcodon s. str., and they are difficult to separate from Sarcodon sometimes.However, morphologically, except for the lack of a distinct smell, bluish or greenish colors in the stipe base and smaller basidiospores can separate members in this group from Sarcodon s.str.Phylogenetically, the monophyly of this group is also strongly supported.Although it seemes that subg.Scabrosum is a distinct group different from Sarcodon and typical Hydnellum, treating it as a distinct genus would make the remaining Hydnellum paraphyletic.The other three edible Hydnellum species in China, H. subalpinum, H. martioflavum and H. versipelle, are distant from those in subgenus Scabrosum, and they are placed in three different clades in the present analysis.Further studies based on more samples might further change the circumscription of Hydnellum and its infrageneric classification in the future.

CONCLUSION
In this study, species diversity of the traditional Hydnellum and Sarcodon species marketed in Southwestern China was analyzed based on morphological and molecular evidence (ITS, nLSU and RPB2).Species diversity of Sarcodon and Hydnellum in the markets is much higher than previously thought, and 17 phylogenetic species are recovered in the present analyses.Eight new species of Hydnellum and Sarcodon, and three new Chinese records of Hydnellum are added to the list of edible stipitate hydnoid fungi in China.Furthermore, Neosarcodon is formally established as a genus, and the generic circumscription of Sarcodon is revised based on the combined morphological and molecular evidence.

Fig. 7
Fig. 7 Basidiomata of the two most common Sarcodon species in markets.a-c Sarcodon imbricatus d-f S. leucopus

Table 1
Specimens sequenced or downloaded from GenBank.Sequences generated for this study are marked in bold

Table 1
(continued) Fig. 2 Maximum likelihood tree showing the phylogeny of Hydnellum, Sarcodon and Neosarcodon based on the combined nLSU, ITS and RPB2 dataset.Branches are labeled with maximum likelihood bootstrap support greater than 70% and Bayesian posterior probabilities greater than 0.95.New species are in bold.★ indicates the Sarcodon species sold in Southwest China, and ▲ means the Hydnellum species sold in Southwest China
: H. versipelle, known from northern portions of the United States and Europe, was found to be sold mixed with H. subalpinum in Sichuan province, where they were called "Jia Hu Zhang Jun".The morphological characteristics of Chinese materials matched those of materials collected from the southeastern United States; and only one different base was observed between the ITS sequences of Chinese and European collections (MK602770, MK602771 and MK602772).